[0001] The present invention relates to novel monoclonal antibodies and, inter alia, an
immunological method utilizing the same. More particularly, the invention relates
to monoclonal antibodies derived from human-human hybridomas capable of recognizing
a differentiated antibody of human lymphocytes and/or exhibiting immunosuppression
against human lymphocytes and free from any adverse side effects when a human body
is administered therewith, immunosuppressive agents with the same as the effective
ingredients, as well as diagnostic methods utilizing the same.
[0002] Along with the rapid progress in the biotechnology in recent years, monoclonal antibodies
are highlighted and many intensive and extensive investigations are being undertaken
in a variety of fields in relation to monoclonal antibodies.
[0003] As is well known, every living body of animal is provided with a function of self-defence
called immunity which is brought to exhibition against a foreign body such as bacteria,
referred to as an antigen, intruding the living body so that the lymphocytes in the
body produce an antibody having an activity of eliminating or holding down the foreign
body out of or in the living body. Under such a situation, lymphocytes of a single
clone characteristically produce a single kind of the antibody which is called a monoclonal
antibody.
[0004] Active investigations are now under way for the application of monoclonal antibodies
in a variety of fields including biochemical analysis such as affinity chromatography,
clinical diagnosis and therapy in the medical science and so on. The application of
monoclonal antibodies in the clinical therapy is exemplified by:
(1) the so-called missile therapeutic method of cancer in which a monoclonal antibody,
of which the antigen is the specific glycoprotein contained in the surface layer of
the cancer cells, is prepared and administrated to a patient of cancer as combined
with an anticancer agent or an anticancer toxin capable of killing the cancer cells
so that the monoclonal antibody selectively adheres to the surface alone of the cancer
cells by the antigen-antibody reaction to have the cancer cells destroyed by the anticancer
agent or toxin connected to the antibody;
(2) the use of a monoclonal antibody in place of conventional immunosuppressive agents
administrated to a patient of organ transplantation with an object ot mitigate the
problem of rejection against the transplanted organ when the major histocompatible
antigens are not in consistency with each other while conventional immunosuppressive
agents sometimes lead to death of the patient due to heavy side effects or infectious
diseases; and
(3) the use of a monoclonal antibody by utilizing the features of high specificity,
stability of quality and low costs inf manufacture in place of conventional antiserums
in the so-called gamma-globulin method in which an antibody called an antiserum prepared
from human blood is administrated to a patient suffering various kinds of immunodeficiency
syndromes or serious infectious diseases.
[0005] Several methods are known and practiced in the prior art for the preparation of a
monoclonal antibody including a method in which an antibody-producing cell and a myeloma
cell are subjected to cell fusion and the thus obtained hybridoma is cultured, a method
in which lymphocytes are subjected to transformation with a virus and the thus obtained
antibody-producing cell strain is cultured, and so on.
[0006] Needless to say, it is essential in the preparation of a human-type monoclonal antibody
to obtain a human antibody-producing cell sensitized by a certian antigen and having
specificity to the same antigen while in vivo stimulation with an antigen is generally
not permitted in human bodies with a few exceptional cases. This situation is the
reason for the unestablished practical technology for the above mentioned possible
application of monoclonal antibodies against various kinds of antigens in general.
An attempt has also been made to obtain a monoclonal antibody by means of a permanent
cell of human obtained by an immortalizing means of a human antibody-producing cell,
such as transformation by the Epstein-Barr virus (EBV), or a human hybridoma obtained
by the cell fusion with a human myeloma cell. Different from the system of mice, unfortunately,
no transforming cells useful as a parent strain have yet been established and no hybridoma
having a stable activity for antibody production has yet been obtained in the system
of human. Accordingly, the monoclonal antibodies available at present are limited
to those derived from mice. Such a monoclonal antibody of mouse-origin may of course
be useful in certian applications, for example, in the biochemical analysis, e.g.,
affinity chromatography, and clinical diagnosis. When the object of application of
such a monoclonal antibody is a therapeutic treatment of patients of certain diseases,
however, the monoclonal antibody is recognized by the human body administrated therewith
as a foreign protein possibly to cause an allergic reaction so that the application
of such a monoclonal antibody to a therapeutic treatment of patients is necessarily
under limitation. It is therefore eagerly desired to develop a technology which provides
a means to conveniently prepare a human monoclonal antibody applicable to a thereapeutic
purpose.
[0007] As to a monoclonal antibody capable of recognizing a differentiated antigen of human
lymphocytes and useful in thereapeutic treatments, no antibody derived from a human-human
hybridoma has yet been disclosed although several mouse-origin ones are known.
[0008] As to a monoclonal antibody used as an immunosuppressive agent, on the other hand,
OKT3 is known and practically used, mainly, in the United States with a purpose of
preventing the rejection reaction against transplanted tissues (see, for example,
Japanese Patent Kokai 55-145617). This antibody, however, is very defective because
it is derived from mouse and acts as a foreign protein in human body to cause serious
side effects when it is administrated to a patient more than once (see Nikkei Biotech,
July 14 issue, 1986, and February 23 issue, 1987). Reportedly, patients administrated
therewith in clinical cases suffered chill, shaking chill and pyrexia after 30 minutes
from administration and a half of the patients fell into dyspnea. What was worse
was that recurrence of the rejection reaction was noted in 12 of 16 cases according
to the report in Rinsho Men-eki (Clinical Immunology), volume 17, No. 10, page 895
(1985).
SUMMARY OF THE INVENTION
[0009] The present invention accordingly has an object, in view of the above described situation
relative to the monoclonal antibodies, to provide a novel monoclonal antibody derived
from a human-human hybridoma capable of exhibiting an activity fo recognizing a differentiation
antigen of human lymphocytes or immunosuppressive effect against human lymphocytes
and useful as a side effect-free therapeutic medicament of human diseases.
[0010] Another object of the invention is to provide a novel immunosuppressive method by
utilizing a monoclonal antibody derived from a human-human hybridoma and administrable
to human without the danger of side effects.
[0011] A further object of the invention is to provide a novel diagnostic method by utilizing
a monoclonal antibody derived from a human-human hybridoma and capable of recognizing
a differentiation antigen of human lymphocytes.
[0012] The above mentioned human-human hybridoma can be obtained by subjecting a transformed
human cell to a teatment to inhibit the cell proliferation and then subjecting the
thus treated transformed human cell to cell fusion with a human antibody-producing
cell.
[0013] The above mentioned diagnostic method is performed by bringing a biological sample
under testing into contact with the above defined monoclonal antibody so as to form
a complex between the monoclonal antibody and the lymphocyte in the sample having
a differentiated antigen and detecting or determining the thus formed complex.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0014] The inventor has conducted extensive investigations to prepare a human-human hybridoma
having a stable activity for producing a human monoclonal antibody and previously
disclosed in Japanese Patent Kokai 63-17688 that such a human-human hybridoma can
be obtained by subjecting a transformed human cell to a treatment to inhibit the cell
proliferation and then subjecting the thus treated transformed human cell to cell
fusion with a human antibody-producing cells.
[0015] Further continued investigations have led to a novel discovery that the above mentioned
human-human hybridoma produces a human monoclonal antibody capable of recognizing
a differentiation antigen of human lymphocytes or exhibiting an immunosuppressive
effect against human lymphocytes, that the thus obtained human monoclonal antibody
can be used as an immunosuppressive agent without the danger of any side effect and
that the human monoclonal antibody can be used in the detection or determination of
human lymphocytes exhibiting a differentiation antigen by forming a complex between
the monoclonal antibody and the lymphocytes.
[0016] The first step in the preparation of a human-human hybridoma having a stable antibody-producing
activity is a treatment of a transformed human cell to inhibit the cell profiferation.
Usable transformed human cells as the parent cell include, for example, human B cells,
human hepatocytes, human splenocytes, tonsillar cells, human lymph node cells and
the like, of which human B cells as transformed are particularly preferable. The
transformation can be performed by a conventional method such as infection of the
cell with a virus and a genetic recombination method.
[0017] The method for inhibiting the proliferation of these transformed human cells includes
chemical treatment, irradiation with high-energy radiation and the like. Preferably,
the treatment to inhibit the proliferation of transformed human cells is performed
by a chemical treatment with a reagent capable of inhibiting synthesis of protein
or RNA such as emetine, actinomycin D and the like either singly or as a combination
of two kinds or more. The amount of the chemical reagent to be used should be appropriately
selected depending on the type of the cells to be subjected to cell fusion. As a rough
measure, the amount of the reagent should be sufficient to completely kill the transformed
human cells within 7 to 10 days. The transformed human cells after the treatment to
inhibit the cell proliferation can be heterozygous or homozygous but homozygous ones
are preferred in respect of the efficiency in the subsequent screening.
[0018] The next step is cell fusion between the transformed human cells after the treatment
to inhibit the cell proliferation in the above described manner and antibody-producing
human cells to produce a human-human hybridoma. Examples of the above mentioned antibody-producing
cell include, for example, human B cells, human plasma cells, human hepatocytes,
human embryonal hepatocytes, human splenocytes, human lymph node cells, cultured human
B cells and the like, of which human B cells and cultured human B cells are preferred.
The antibody-producing human cells can be used as isolated from a tissue or blood
of human body but it is optional to use the same after a transformation treatment.
It is generally desirable here that the cell fusion is followed by a treatment with
an anti-HLA antibody to exclude the non-fusion antibody-producing human cells. The
cell fusion is performed by a known method such as the method utilizing polyethylene
glycol, virus and the like and the electric cell fusion method. Preferably, the cell
fusion is performed by using the proliferation-inhibited human cells of 1 to 30 times
in number based on the antibody-producing human cells. Various types of culture media
can be used for the purpose of cell fusion including, for example, RPMI 1640 culture
medium, MEM culture medium and modified Dulbecco's culture medium as well as serum-added
and serum-free culture media thereof.
[0019] The human-human hybridoma obtained in this manner is then subjected to inspection
of the desired antibody-producing activity and cloning.
[0020] The antibody-producing activity can be checked by the assay for the antibody produced
by the human-human hybridoma. This antibody assay is performed by testing the reactivity
thereof against a sensitized antigen according to one of various known methods for
the detection of antibodies in general such as ELISA method described in Meth. of
Enzymol., volume 70, pages 419 to 439 (1980), plaque method, spot method, agglutination
reaction method, Ouchterlony method, enzyme immunoassay (EIA), radioimmunoassay (RIA),
cytotoxicity assay and the like (see Hybridoma Method and Monoclonal Antibody, published
by R & D Planning Co., pages 30 to 53, 1982, and elsewhere). The cloning can be easily
performed by repeating the known step of sub-culture or by undertaking a conventional
limiting dilution method.
[0021] The human-human hybridoma produced in this manner is cultured usually in a suitably
culture medium such as RPMI 1640 culture medium, MEM culture medium and modified Dulbecco's
culture medium as well as serum-added and serum-free culture media thereof at a temperature
of 36.5 to 37.5 °C with a pH of the medium controlled in the range of 6.7 to 7.5 or,
preferably, 7.0 to 7.3 and a concentration of dissolved oxygen of 0.5 to 6 ppm or,
preferably, 3 to 5 ppm. When the pH of the culture medium is outside the above mentioned
range, a retarding effect is caused on the rate of hybridoma multiplication, especially,
in the initial stage. When the concentration of the dissolved oxygen is outside the
above mentioned rage, an adverse effect is caused on the productivity of the desired
antibody by the hybridoma. The vessel used for culturing is not particularly limitative
including microplates, Petri dishes, glass flasks, culture tanks and the like depending
on the desired scale of culturing.
[0022] The human hybridoma cultured in this manner produces the desired human-type monoclonal
antibody and secretes the same in the supernatant of the culture medium. The human
monoclonal antibody can be isolated and purified from the culture medium by one or
a combination of conventional methods including centrifugal separation, ultrafiltration,
gel filtration, ion-exchange chromatography, affinity chromatography and the like.
In particular, a good efficiency is obtained in the isolation and purification thereof
by the method of affinity chromatography using a column loaded with protein A.
[0023] Being derived from a human-human hybridoma, the monoclonal antibody obtained in
this manner can be used as an immunosuppressive agent for human without being accompanied
by any side effect.
[0024] When the human-human hybridoma is produced by appropriately selecting the transformed
human cell and the human antibody-producing cell used in the cell fusion, the human-human
hybridoma can secrete a human monoclonal antibody capable of recognizing a differentiation
antigen of human lymphocytes or exhibiting an immunosuppressive effect against human
lymphocytes. An example of such a human-human hybridoma is the 223.10.33 strain deposited
at Fermentation Research Institute, Japan, with a deposit No. FERM BP-2384, which
has been obtained by the cell fusion between the SA-1 strain, the transformed human
B blast cells as the transformed human cells, and the EBV-SU strain, the transformed
human antibody-producing B cells as the human antibody-producing cells. The human
monoclonal antibody here obtained is novel in respect of the capacity of recognizing
a differentiation antigen against human lymphocytes or the immunosuppressive effect
against human lymphocytes and is useful in the therapeutic and diagnostic purposes,
in particular, as an immunosuppressive agent without the trouble of any side effect
on human body.
[0025] The medicament form in which the inventive human monoclonal antibody is used as
an immunosuppressive agent is not particularly limitative including pills, tablets,
granules, powders, capsules, syrups, ointments, liniments, injections and the like.
It is of course that each medicament form is prepare dby compounding the monoclonal
antibody as the effective ingredient with any pharmacologically acceptable additives
conventionally used in medicament preparations.
[0026] As to the dose of the medicament to be administrated, it is usual that a sufficient
effect as desired can be obtained with 1 mg to 5 g of the administrated medicament
calculated as the monoclonal antibody per day per adult.
[0027] The monoclonal antibody of the invention has no reactivity with normal T and B cells
of human peripheral blood but is reactive with the lymphocytes stimulated with various
kinds of mitogens and interleukin-2 (IL-2). Therefore, the monoclonal antibody may
provide a means to recognize appearance of a differentiation antigen by the activation
of the lymphocytes and is useful in the diagnostic purpose of certain diseases. Namely,
a differentiation antigen by the activation of human lymphocytes appears to recognize
and eliminate the foreign body by the activity of the immune system when a human body
is infected with bacteria or virus or when a human body is suffering a suppurative
disease. Since the monoclonal antibody of the invention is capable of recognizing
such a human lymphocyte activated and exhibiting a differentiation antigen, the monoclonal
antibody can be effectively utilized in an
in vivo or
in vitro diagnostic purpose of a patient of infectious diseases or after a disease or operation
by bringing the biological sample into contact with the monoclonal antibody to form
a complex with the lymphocytes and detecting or determining the complex-forming lymphocytes.
[0028] As is understood from the above given description, the monoclonal antibody of the
invention capable of recognizing a differentiated antigen of human lymphocytes is
absolutely free from the danger of any side effects when it is administrated to a
human body since it is a product of a human-human hybridoma and not a foreign protein
against the human body. Accordingly, application of the monoclonal antibody is not
limited to an effective ingredient in an immunosuppressive agent but it can be effectively
used widely in the therapeutic and diagnostic fields.
[0029] As to the application as an immunosuppressive agent, the monoclonal antibody of the
invention as the principal ingredient of an immunosuppressive agent can be used satisfactorily
as a rejection-preventing agent in an operation of organ transplantation or as a therapeutic
medicament of certain immuno-abnormalities such as autoimmune diseases because, different
from conventional immunosuppressive agents such as OKT3 and the like derived from
mice, it is derived from a human-human hybridoma and absolutely free from the disadvantage
of side effects when it is administrated to a human body.
[0030] The monoclonal antibody of the invention can recognize an antigen having a molecular
weight in the range of 100 to 105 kDa, 120 to 130 kDa and 270 to 280 kDa for which
no detecting means was hitherto known so that the inventive monoclonal antibody may
provide a possibility of detecting an antigen which cannot be detected in the prior
art.
[0031] In the following, examples are given to illustrate the invention in more detail but
not to limit the scope of the present invention an any way.
Preparation Example. Preparation of a human-human hybridoma 223.10.33 strain
(I) Preparation of antibody-producing B cells
[0032] Peripheral blood was taken from pregnant volunteer (SU: HLA-A2, -: Bw 46.22: Cw 7,
-: DR 4,8) and the lymphocytes were collected therefrom by the Conray-Ficoll gradient
method. The B cells were obtained by separating the T and B cells by the rosette formation
method using SRBC treated with neuraminidase.
(II) Sensitization of B cells with Epstein-Barr virus (EBV)
[0033] An EBV strain was prepared in a culture of EBV-transformed marmoset cells, B95-8
strain, growing in a RPMI 1640 culture medium with addition of 10% of fetal calf serum
(FCS) and the supernatant containing the viruses was twice filtered. The virus strain
was added to 100 µl of a suspension containing the B cells in a density of 5 × 10⁴
cells/100 µl and incubated at 37 °C for 1 hour. Thereafter, a 800 µl fresh portion
of the same 10% FCS-added RPMI 1640 culture medium was added thereto. The incubation
was continued by adding the same volume of the fresh culture medium in every 3 to
4 days. Transformed B cells, referred to as the EBV-SU hereinbelow, could be detected
about 2 weeks after sensitization.
(III) Treatment of parent cells, EBV-transformed B lymphoblast cells (SA-1), with
emetine and actinomycin D
[0034] Homozygous SA-1 cells (HLA-A24, B7, C-, DR 1, Dw 1) were cultured in a 10% FCS-added
RPMI 1640 culture medium. The cells harvested at the growing phase were washed by
centrifugation at 1000 rpm three times each for 10 minutes with the RPMI 1640 medium
and conditioned in a density of 1 × 10⁴ cells/ml followed by a treatment with an emetine
hydrochloride solution of 5 × 10⁻⁵ M concentration and 0.1 µg/ml of actinomycin D
at 37 °C for 2 hours. The above mentioned concentrations of emetine and actinomycin
D were sufficient to completely suppress division and proliferation of the SA-1 cells.
free emetine and actinomycin D were completely removed from the cells by washing three
times with fresh portions of the RPMI 1640 medium to give proliferation-inhibited
SA-1 cells.
(IV) Cell fusion and growth conditions for hybrid
[0035] The EBV-SU cells collected and washed three times with the RPMI 1640 medium were
suspended in the RPMI 1640 medium and mixed with the proliferation-inhibited SA-1
cells in a proportion of 10: 1.
[0036] The above prepared cell mixture was admixed with 0.25 ml of a 15% DMSO-42.5% polyethylene
glycol (PEG1000) solution taking 1 minute under continuous swilling and then with
a 9 ml fresh portion of the RPMI 1640 medium warmed at 37 °C at a rate of 1 ml/minute.
[0037] The cells in suspension were consolidated by centrifugation at 1000 rpm for 10 minutes
and then suspended in the 10% FCS-added RPMI 1640 medium. The suspension was distributed
to the wells of microtiter plates while the density of the cells in the suspension
was adjusted such that 5 × 10⁴ cells were distributed on each well. An additional
0.1 ml fresh portion of the culture medium was added to each well in every 2 to 3
days.
(V) Detection of HLA antigen and selection of fused cells
[0038] The surface HLA-DR antigen of the hybridoma was detected by the microlymphocytotoxicity
test to conduct selection of the fused cells having the 1, 4 and 8 antigens. Two hybridomas
having the HLA-DR 1, 4 and 8 antigens were obtained from a million of the EBV-SU cells.
Further, the desired hybridoma was obtained by the limiting dilution method.
[0039] In the next place, this hybridoma 223 was subjected to cloning in a density of 10
cells/well to give a subclone 223.10.33 strain.
Example 1. Preparation of immunoglobulin M (IgM) by the 223. 10. 33 strain in serum-added culture
medium
[0040] The cells of the 223. 10. 33 strain obtained in the Preparation Example were subcultured
in a 10% FCS-added RPMI 1640 culture medium contained in a flask at 37 °C under an
atmosphere of a gaseous mixture of 5% carbon dioxide and 95% air. The thus subcultured
cells were dispersed in a 5 liters fresh portion of the same culture medium in a cell
density of 2 × 10⁵ cells/ml and agitation culture was performed for about 6 days in
a spinner flask of 5 liters capacity rotated at 20 rpm with a concentration of dissolved
oxygen of 4 ppm to give a culture containing about 1 × 10⁶ cells/ml. The cells were
removed from the culture by centrifuging at 4000 rpm for 5 minutes to give 5 liters
of the supernatant which was subjected to ultrafiltration using a membrane capable
of eliminating particles having a molecular weight smaller than about 300,000 to
reduce the volume to 500 ml. Ammonium sulfate was added to the thus concentrated
supernatant in a concentration of 50% saturation and the mixture was centrifuged to
collect the precipitates as an immunoglobulin (IgM) -containing crude product. This
crude product was dissolved in 150 ml of a 10 mM phosphate buffer solution having
a pH of 7.4 and containing 0.9% of sodium chloride. The solution was dialyzed against
the same buffer solution for about 18 hours to give about 200 ml of a dialyzed solution.
A chromatographic column having an inner diameter of 5 cm and a height of 90 cm filled
with Sephacryl S-300 was loaded with 100 ml of the thus obtained dialyzed solution
and elution was performed with the same buffer solution. Determination of IgM in the
eluate fractions was undertaken by the photometric measurement at a wavelength of
280 nm and IgM determination by the ELISA method. The IgM-containing fraction in a
volume of about 250 ml was subjected to ultrafiltration to give a concentrated solution
in a volume of 6 ml containing an antibody against the 223. 10. 33 strain. This antibody
solution contained 1 mg/ml of the IgM and the titer thereof was 256 times by the complement-dependent
cellular cytotoxic assay.
Example 2. Preparation of IgM by the 223. 10. 33 strain in GIT culture medium
[0041] The culture medium used here was a commercially available GIT culture medium admixed
with a cell growth factor in an animal serum in place of the fetal calf serum.
[0042] The cells of the 223. 10. 33 strain obtained in the Preparation Example were subcultured
in the above mentioned culture medium contained in a flask at 37 °C under an atmosphere
of a gaseous mixture of 5% carbon dioxide and 95% air. The thus subcultured cells
collected by centrifugation at 1500 rpm for 5 minutes were added to 20 flasks of each
75 cm² size containing a 30 ml fresh portion of the GIT culture medium and suspended
therein in a density fo 4. 6 × 10⁵ cells/ml. The suspensions in the 20 flasks were
combined to make up a volume of 600 ml and introduced into a spinner flask of 2 liters
capacity together with a 1400 ml fresh portion of the same culture medium to give
a cell density of 2 × 10⁵ cells/ml as dispersed followed by further continued agitation
culture at 32 rpm with a concentration of dissolved oxygen of 4 ppm.
[0043] When the cell density had reached 1 × 10⁶ cells/ml after 6 days of culturing, the
cells were removed from the culture by centrifugation at 4000 rpm for 5 minutes to
give 2 liters of the supernatant. The supernatant was passed down through a chromatographic
column of 0.5 cm inner diameter and 15 cm length filled with Sepharose CL-6B loaded
with protein A at a flow rate of 1 ml/minute to have the IgM adsorbed on the filler
followed by washing with a 0.02 M phosphate buffer solution having a pH of 4 at a
flow rate of 1 ml/minute and then elution of the IgM with a 0.03 M phosphate buffer
solution having a pH of 3 at a flow rate of 1 ml/minute. Detection of the IgM in the
eluate fractions was undertaken by the photometric measurement at a wavelength of
280 nm and determination of IgM by the ELISA method.
[0044] Gel-filtration chromatography by FPLC was undertaken for 200 ml of the thus obtained
IgM-containing fraction through a Pharmacia Superose 6 column (a product by Pharmacia
Co.) using a 0.01 M phosphate buffer solution having a pH of 6. 5 at a flow rate of
0. 5 ml/minute as the eluant and the IgM-containing fraction in a volume of 100 ml
was subjected to ultrafiltration using a membrane capable of eliminating particles
having a molecular weight smaller than about 300,000 to give 10 ml of a concentrated
solution containing an antibody against the 223. 10. 33 strain corresponding to 10
mg of the IgM. This antibody was identified to be pure by the result of the SDS electrophoresis.
The titer of this antibody solution was 512 times as determined by the complement-dependent
cytotoxicity assay.
Example 3. Reactivity of the 223. 10. 33 antibody with mononuclear cells fo peripheral blood:
complement-dependent cytotoxicity assay
[0046] The peripheral blood samples taken from 18 normal persons were subjected to the
Conray-Ficoll method to isolate the mononuclear cells which were further separated
into the T cells and B cells by the nylon column method and sheep red cells rosette
formation method. Experiments were conducted by using these T and B cells as well
as the peripheral blood mononuclear cells stimulated with three kinds of mitogens
including PHA, i.e. phytohemagglutinin, PWM, i.e. pokeweed mitogen, and ConA, i.e.
concanavalin A, and IL-2, i.e. interleukin 2. Thus, the PHA, PWM, ConA and IL-2 were
added each to a RPMI 1640 culture medium containing 10% of human serum in a concentration
of 0.1%, 1%, 10 µg/ml and 1 u, respectively, and cultured for 5 days to effect stimulation.
A 1 µl portion of the antibody solution prepared in Example 1 was added to a Terasaki
plate and the above described target cells wwere poured portionwise to each of the
wells to give 2000 cells/GVB⁺⁺ with a gelatin-Veronal ⁻buffer solution and the reaction
was effected at 37 °C for 1 hour. Thereafter, 5 µl of a twice-diluted rabbit complement
(a product by Peritus Co.) were added thereto to effect the reaction for 2 hours at
room temperature followed by eosin stain and formalin fixation to microscopically
examine life or death of the cells. The results of the reactivity as given by the
ratio of the number of positive cells to the total number of the examined cells were
as follows.
Peripheral blood T cells: |
0/18 |
Peripheral blood B cells: |
0/18 |
PHA-stimulated peripheral blood mononuclear cells: |
5/5 |
PWM-stimulated peripheral blood mononuclear cells: |
5/5 |
ConA-stimulated peripheral blood mononuclear cells: |
3/5 |
IL-2-stimulated peripheral blood mononuclear cells: |
5/5 |
[0047] The above given results indicate that the 223.10.33 antibody was reactive with the
peripheral blood mononuclear cells stimulated with the mitogens or IL-2 but non-reactive
with the unstimulated peripheral blood T cells and B cells.
Example 4. Reactivity of the monoclonal antibody with activated human T cells
[0048] The peripheral blood smaples taken from 3 normal persons were subjected to the Conray-Ficoll
method to isolate the mononuclear cells from which the T cells were separated by the
sheep red cells rosette formation method. These cells were suspended in a RPMI 1640
culture medium with admixture of 20% of human serum and containing 10 u or 1 u of
IL-2 in a cell density of 1 × 10⁵ cells/ml and cultured at 37 °C under an atmosphere
of a gaseous mixture of 5% carbon dioxide and 95% air with periodical replacement
of a half volume of the culture medium once in every three days. The thus cultured
cells were collected from each culture medium after 1, 2, 3, 5 and 7 days fo culturing
as well as before the start of culturing and microscopically examined for life or
death of the cells by the cytotoxicity assay in the same manner as in Example 3 to
give the results shown in Table 1 below by the percentage of the injured T cells on
an average for the 3 persons. As is shown in the table, the reactivity of the 223.
10. 33 antibody was apparent after 5 days of culturing and later on.
Table 1
IL-2 stimulant concentration |
Days of culturing |
|
0 |
1 |
2 |
3 |
5 |
7 |
10 u |
0 |
0 |
10 |
20 |
80 |
60 |
1 u |
0 |
0 |
20 |
10 |
50 |
30 |
Example 5. Reactivity of the monoclonal antibody with transformed human B cells
[0049] The peripheral blood samples taken from 3 normal persons were subjected to the Conray-Ficoll
method to isolate the mononuclear cells from which the B cells were separated by the
sheep red cells rosette formation method. These B cells were transformed with the
Epstein-Barr virus to give EBV-B cells according to the procedure described in Nature,
volume 269, pages 420-422 (1977). The reactivity of the antibody with these cells
was examined in the same manner as in Example 3 by continuing the culturing up to
34 days to give the results that the antibody was non-reactive with the cells until
9 days of culturing but reactive after 12 or 14 days of culturing to give the percentages
of the injured cells shown in Table 2 below.
Table 2
Days of culturing |
9 |
12 |
14 |
16 |
19 |
21 |
24 |
34 |
% injured cells, average |
0 |
20 |
50 |
50 |
70 |
50 |
70 |
80 |
Example 6. Influence on the antibody production of mononuclear cells
[0050] Mononuclear cells were isolated from normal human peripheral blood by the Conray-Ficoll
method. The cells were suspended in a RPMI 1640 culture medium containing 1% of PWM
and admixed with 10% of human serum in a density of 5 × 10⁵ cells/ml and cultured
at 37 °C using a 96-well U-shaped microplate under an atmosphere of a gaseous mixture
of 5% carbon dioxide and 95% air. At the moments of 1, 3, 6, 12, 24, 48, 72, 96 and
120 hours from the start of culturing, a 20 µl portion of an antibody solution dissolved
in the RPMI 1640 culture medium was added to each well. The antibody solution was
a 5 mg/ml solution of the 223.10.33 antibody or a 5 mg/ml solution of the standard
human IgM supplied by Kappel Co. After a week of continued culturing, the cells were
recovered from the culture by centrifugation and subjected to detection of the IgC-reactive
and IgM-reactive plaque-forming cells (PFC) by the reverse plaque method descirbed
in "Manual of Immunological Experiments", C, pages 2003-2004 to give the results shown
in Table 3, in which the numbers of the IgG-reactive and IgM-reactive PFCs (IgG-PFC
and IgM-PFC) are given per 10⁴ of the total cells. These results indicate that the
223.10.33 antibody clearly had an activity of inhibiting production of IgG and IgM
throughout the period from immediately after stimulation with PWM to the stage of
differentiation and proliferation of the cells while the commercially available human
IgM had no such an activity.
Table 3
PWM |
Culturing time up to addition of antibody, hours |
IgG-PFC/10⁴ |
IgM-PFC/10⁴ |
|
|
223.10.33 antibody added |
Human IgM added |
223.10.33 antibody added |
Human IgM added |
No |
|
0 |
0 |
0 |
0 |
Yes |
|
171 |
171 |
266 |
266 |
Yes |
0 |
0 |
134 |
0 |
230 |
Yes |
1 |
0 |
81 |
0 |
177 |
Yes |
3 |
0 |
281 |
0 |
499 |
Yes |
6 |
0 |
135 |
0 |
310 |
Yes |
12 |
0 |
132 |
0 |
243 |
Yes |
24 |
0 |
117 |
0 |
227 |
Yes |
48 |
0 |
215 |
18 |
358 |
Yes |
72 |
5 |
141 |
2 |
258 |
Yes |
96 |
0 |
200 |
5 |
276 |
Yes |
120 |
0 |
172 |
0 |
327 |
Example 7. Assay for the molecular weight of recognized antigens
[0051] The Western-blot technique was applied to the assay of the molecular weight of the
recognized antigens. Thus, B-85 cells, i.e. B lymphoblast-like cell line transformed
with EBV, and BT-1 cells, i.e. Burkitt lymphoma, having particularly high reactivity
with the 223.10.33 antibody were solubilized with 0.5% NP-40 and nuclei were removed
by centrifugation to give a fraction containing the cell membranes. This fraction
was subjected to electrophoresis with an SDS polyacrylamide gel. Thereafter, transcription
was undertaken from the gel to a nitrocellulose filter followed by the reaction with
the 223.10.33 antibody to detect the recognized antigens using a goat IgM labelled
with peroxidase (POD) (a product by Kappel Co.). As a result, three developed bands
were detected respectively corresponding to the molecular weights of 100 to 105 kDa,
120 to 130 kDa and 270 to 280 kDa.
Example 8. B cell marker detectable with the monoclonal antibody
[0052] Reactivity of the 223.10.33 antibody was examined using strains of B cells at different
stages of differentiation as the target cells by the indirect fluorescent antibody
technique described in Handbook in Clinical Immunology, page 1510 (1984).
[0053] The CD antibodies [see Clinical Immunology, volume 18, Summerly Special Issue, page
149 (1986)] used here were B 2 (CD 21, coal tar clone), Leu-14 (CD 22, a product by
Fujisawa Yakuhin Co.) and H 107 (CD 23, a product by Nitiray Co.). The results are
shown in Table 4, from which it is confirmed that the 223.10.33 abtibody is closely
correlated with the CD 23 antibody.
Table 4
Differentiation stages and cell strains |
Pre-B cells |
B lymphocytes |
plasma cells |
Monoclonal antibody |
|
|
immature/resting |
mature/activated |
|
|
|
Ramos |
Daudi |
BALL-1 |
RAJI MGAR SPACHEO MZ070782 PF97387 |
AKIBA RSH OLL HID |
BM16 Sa-IV AMALA EK/OH |
KT-12 COX SP-0010 RML |
B-85 BT-1 MANN |
RPMI8226 |
B2 EBV recepter |
- |
+ |
+ |
+ |
+ |
| |
- |
- |
- |
(CD21) C3D recepter |
|
|
|
|
|
|
|
|
|
Leu-14 |
+ |
+ |
- |
+ |
+ |
+ |
+ |
- |
- |
(CD22) |
|
|
|
|
|
|
|
|
|
H107 Fc recepter |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
+ |
(CD23) |
|
|
|
|
|
|
|
|
|
223.10.33 |
- |
- |
- |
+ |
+ |
+ |
+ |
+ |
- |
Note: +: positive, -: negative, |: false positive |